PROJECT SUMMARY/ABSTRACT Our proposed studies will develop a novel high throughput microfluidic platform to generate microenvironmental oxygen and hydrogen sulfide landscapes allowing investigators to study real-time hypoxic and H2S signaling. If successful, these studies could define novel protective mechanisms mediated by hypoxic signaling or the gasotransmitter H2S and identify precise therapeutic windows which optimize vascular health. Precise spatial and temporal control of dissolved gases is a major limitation for current studies that rely on chemical donors for gasotransmitters or large volume chambers that do not enable the study of real-time interactions between cells exposed to varying gradients of dissolved gases. The insights gained into the interactions between hypoxic and normoxic cells or cells exposed to varying levels of H2S will allow us and other researchers who will use this device study cellular cross-talk. Importantly, studying the distinct thresholds of signaling will allow researchers to define the therapeutic windows at which dissolved gases or their downstream signals induce maximal protection and avoid deleterious effects. The combination of the high-throughput design with precisely defined landscapes will greatly facilitate the identification of druggable therapeutic targets for oxygen and H2S signaling pathways. We will use 3D printing to develop 96-well plate culture gas and fluidic networks, bond gas permeable microfluidic networks to the base of the pillars, and control synergistic dissolved gas landscapes hypoxia and hydrogen sulfide. The 96 well plate will be used to study biological insights into hypoxic and H2S crosstalk. In Aim 1, we will develop a high-throughput microfluidic dissolved gas landscape platform by a) developing, fabricating and validating linear gradient and oscillating dissolved gas (O2 and H2S) landscapes in a 3D printed 96 well plate insert b) integrating a standalone gas blender into the top of the insert to generate multiple dissolved gas conditions c) integrating plate to plate gas connections to facilitate stacking for higher throughput experimentation and fluidic channels for media exchange to facilitate long term cultures. In Aim 2, we will use the platform to study cellular responses of vascular endothelial cells to dynamic dissolved gas landscapes to determine the effects of precisely controlled O2 and H2S landscapes which enable monitoring real-time interactions between cells exposed to varying levels of O2 and H2S on the proliferation, angiogenic capacity, formation of reactive oxygen species and adaptive autophagy of primary human endothelial cells.